Genes

1
Genes Development

• Part 2 Molecular Methodology Proves Differential
  Gene Expression

2
Differential Gene Expression

• Demonstration of differential gene expression has
  been done through a variety of methods
• Cytogenetics in Drosophila
• Polytene chromosomes
• RNA hybridization competition Rot curves
• RNA localization
• Biochemical Immunological techniques

3
Differential Gene Expression

• Polytene chromosomes
• 10 rounds of replication without cell division
• 1024 side-by-side chromatids
• Stained chromosomes seen during interphase
• Puffs
• Regions where polytene chromosome appears much
  wider
• Locations of puffs change during development and
  in different cell types
• Location of transcription
• Implies that transcription occurs in different
  places on chromosomes in different cells

4
Polytene Chromosomes
Fluorescence microscopy of propidium I stained
Drosophila polytene chromosomes (red) overlayed
with immuno- stained chromatin scaffold protein
(yellow). Chromocenter (centromere) indicated
with arrowhead. Puffs indicated by arrows
5
Methods Microscopy

• Low power stereo microscopy (dissecting scope
  4-100X)
• High-power light microscope (20-400X)
• Nemarski interference optics
• Fluorescence microscopy
• Confocal laser microscopy (stereoimage
  3D/optical sectioning)
• Electron microscopy
• Scanning (SEM)
• Photomicroscopy

6
Differential Gene Expression

• RNA hybridization
• Isolate RNA from different cell types
• Make radiolabeled cDNA from one mRNA
• Hybridize labeled cDNA to mRNA
• Compete mRNA-cDNA hybridization by adding excess
  mRNA from other cell type
• Amount of competition indicates number of same
  mRNA species in both cells.
• The presence of mRNA-cDNA hybrids remaining after
  competition with unlabeled mRNA indicates unique
  transcripts

7
Methods RNA hybridization
mRNA from cell type B
mRNA from cell type A
Synthesize1st strand cDNA
All B mRNAs hybridize to cDNAs
Only some type A mRNAs hybridize
8
RNA Localization

• Northern analysis
• Isolate total or pA RNA
• Separate by size with electrophoresis (special
  gel to prevent secondary structure)
• Transfer electrophoresed RNA to
  nitrocellulose/nylon membrane (blot)
• Probe blot with radiolabeled cDNA probe
• Northern analysis requires that cDNA be cloned

9
Northern Analysis Gel
Electrophoresis
Formaldehyde gels Methyl-mercury-OH gels
10
Northern Analysis ElectroBlotting
11
Northern Analysis ElectroBlotting
12
Northern Analysis Probing
13
RNA Localization

• Developmental Northerns
• Temporal expression information
• Spatial expression information (limited to
  microdissected embryo regions and tissues)
• Requires that RNA species being detected be
  fairly abundant (large amounts of RNA must be
  isolated/purified)

14
RNA LocalizationDevelopmental Stages
15
RNA LocalizationDevelopmental Stages
16
RNA Localization in Tissues
17
RNA Expression Following Experimental Treatments
18
RNA Expression Following Experimental Treatments
19
RNA Localization

• RT-PCR
• Isolate RNA (total or pA)
• Reverse transcribe using oligo dT primer
• Amplify cDNA by PCR using gene specific primers
  and using only 10-15 rounds
• Run PCR product on gel (regular agarose)
• Southern blot and probe with cDNA probe
• Much more sensitive than northern blot

20
RNA Localization

• Whole mount in situ hybridization allows us to
  see which cells express a given mRNA.
• Hybridize antisense RNA probe to mRNA in embryo
• Information temporal and spatial expression
  pattern
• Putative function of gene product based on
  expression pattern

21
Methods Whole-mount in situ hybridization
22
Methods Whole mount in situ hybridization
m
FGF8 expression in 3-day chick. Expression
detected in somites (s), limb buds (l), brachial
arches (b) midbrain-hindbrain boundary (m)
b
s
l
l
23
Whole Mount In Situs
24
Whole Mount In Situs
25
Biochemical Immunological Techniques

• Biochemical purification characterization
• Generation of antibodies that specifically
  recognize proteins
• pAb polyclonal antibodies
• Many Abs varieties generated against a protein
• Each Ab may recognize a unique epitope
• mAb moloclonal antibodies
• One Ig producing cell (B-cell) reacts to protein
• One variety of Ab produced which recognizes one
  specific epitope

26
Biochemical Techniques

• Classical protein biochemistry
• Column chromatography
• Preparative electrophoresis
• Amino acid sequencing
• Structural Biochemistry
• X-ray crystallography, CD-ORD, MS-AS, NMR
• Computer generated structure predictions

27
Immunological Techniques

• Immunohistochemistry (whole mount section)
• Immunolocalization
• Western analysis
• Immunoprecipitation IP
• Co-immunoprecipitation coIP
• cDNA expression library screening

28
Western Analysis

• Separate proteins by electrophoresis (SDS-PAGE)
• Incubate with antibody to specific protein and
  detect presence/absence
• IP protein using Ab to first protein
• Run gel and probe with 2nd Ab to second protein
  to determine if two proteins interact

29
Western Analysis
30
Identifying Developmentally Significant Genes

• Differential Screens
• Homology Screens
• Functional Screens
• Require generation of cDNA libraries followed by
  screening for desired clones
• Mutagenic Screens
• Requires an assayable phenotype and viable
  heterozygote (doesnt work well for dominant
  lethal mutations)

31
Differential Screening

• Use of a subtracted probe to screen a tissue
  specific cDNA library
• Subtracted probe is not gene-specific rather it
  is differentiation-state-specific probe
• cDNA made from one tissues mRNA
• cDNA is hybridized to mRNA from another related
  yet different cell type to remove all transcripts
  in common (housekeeping genes)
• Probe has then has common RNA transcripts
  subtracted from it

32
Differential Screening Subtracted Library
Epidermal cells
Neuroblasts Neuroectoderm
pA RNA
pA RNA
1st strand cDNA
Anneal epi RNA neuro cDNA (2X)
recover ss cDNA from hydroxyapatite column
(neuro epi specific cDNAs)
Anneal ss cDNAs from HA column to neuro mrna
recover ds cDNA/mRNA hybrids from HA column
(neuro specific cDNAs)
Use neuro specific cDNAs to make library
33
Differential Screen Subtracted Probe /- Screen
Epidermal cells
Neuroblasts Neuroectoderm
pA RNA
pA RNA
1st strand cDNA (32P-label)
Anneal epi RNA neuro cDNA
recover ss cDNA (HA column) (neuro epi
specific cDNAs)
Anneal ss cDNAs from HA column to unlabeled,neuro
mRNA
recover ds cDNA/mRNA hybrids from HA column
(neuro specific cDNAs)
Use labeled cDNA as - probe to screen library
Use labeled cDNA as probe to screen library
34
Differential Screen Subtracted Probe /- Screen
35
Homology Screens

• Screen a cDNA library for a related gene from a
  different species
• Reduce stringency of hybridization so that to
  compensate for slight differences in the gene
  sequences between the two species
• Find a gene in Drosophila
• antennapedia
• Is there a related gene in mammals?
• Hox genes

36
Functional Screens

• Add back mRNAs to rescue a mutant phenotype
• Drosophila
• Cloning of bicoid by adding back fractions of
  mRNAs from wt eggs to bicoid mutant eggs
• Xenopus
• Cloning of noggin by adding back mRNAs to
  ventralized embryos
• Screening of expression libraries
• Look for functional interactions two hybrid

37
Functional Screen Yeast 2-Hybrid

• Fuse DNA binding domain to bait
• cDNA coding DNA binding domain of yeast txn
  factor
• Ligated in-frame with cDNA coding protein of
  interest bait
• Make cDNA library in vector where cDNAs will be
  in-frame with txn activation domain of yeast txn
  factor
• This is the prey
• If bait and prey proteins interact,
  txn-activation DNA-binding domains of yeast txn
  factor will be next to each other and will drive
  expression of gene in yeast that allows yeast to
  live

38
Functional Screen Yeast 2-Hybrid
Prey
UnkP
Gal4-AD
UnkP
Gal4-AD
Bait
UnkP
Gal4-AD
POI
LexA-DBD
UnkP
Gal4-AD
Transformed into yeast
UnkP
Gal4-AD
UnkP
Gal4-AD
Transform yeast cells expression bait with cDNA
prey library. Each yeast cell will make 1 of the
prey fusion proteins
39
Functional Screen Yeast 2-Hybrid
40
Mutagenic Screens Chemical/Radiation

• Mutagenize one sex (usually male)
• Mate to wt female
• All progeny (F1) are heterozygotes for any
  mutations
• Outcross all F1 females to wt males or backcross
  FI males to mother
• 50 of F2 will be heterozygotes
• Testcross F2 sibs to produce
• 25 with mutant phenotype
• Outcrossed F1 becomes founder for further
  generations to examine and clone mutated gene

41
Mutagenic Screens Insertional Mutagenesis

• Create transgenic animals with heterologous
  marker gene inserted randomly into genome
• LacZ or GFP most common marker gene
• Screen for animals with mutant phenotype
• Identify location of insertion and clone
  surrounding sequences
• Use probe corresponding to marker gene sequences

42
Functional Analysis of Developmental Genes

• Mutant phenotype associated with gene
• Generation of mutant phenotype when not already
  known
• Targeted disruption (transgenic analysis)
• Mis-expression
• Ectopic expression
• Over expression
• Biochemical analysis
• Subcellular location
• Protein-protein interactions
• Enzymology

43
Transgenic Analysis

• Random insertion of transgenes (for mutagenesis)
• Targeted insertion of transgenes
• Knockout
• Knockin
• Requires special vectors
• contains flanking sequences to permit homologous
  recombination between construct and chromosome
• Contains selectable marker to permit survival
  only of homologous recombination and not
  non-homologous

44
Transgenic Analysis
neor
5 flank
3 flank
Vector for homologousrecombination
HSV-tk
45
Transgenic Analysis
Gene of interest
46
Transgenic Analysis
neor
Homologous recombination replaces region of gene
with neomycin resistance gene and disrupts
generation of functional protein. Neor allows for
cells to be selected for using antibiotic
neomycin.
47
Transgenic Analysis
neor
HSV-tk
Non-homologous recombination inserts thymidine
kinase. The presence of gene allows cells
containing it to be killed by the thymidine
analog gancyclovir or FIAU. Only HSV (viral) tk
will phosphorylate the nucleotide analog so only
the cells with HSV-tk will be killed. The
phosphorylated analog stops DNA synthesis when it
is incorporated by DNA polymerase.
48
Transgenic Analysis
iodo
fluoro
arabinose
49
Transgenic Analysis
50
Transgenic Analysis
FIAU insensitivity
51
Transgenic Analysis
52
Transgenic Analysis
53
Functional Analysis by Misexpression

• Ectopic expression
• Express protein in cells which would not normally
  have the protein present
• Mis-expression at different stages which could be
  in normal or ectopic locations
• Drosophila, Xenopus, zebrafish, C. elegans,
  cultured cells (ES cells ? mouse)
• Systems conducive to DNA/RNA injection/transfectio
  n/injfection

54
Misexpression

• Allows expression of wt or mutant forms of
  protein
• Allows knockout using antisense RNA, antisense
  oligos, or dominant-negative proteins
• Allows increase in activity of protein by
  expressing constitutively active proteins

55
Misexpression

• Injection of either synthetic mRNAs or DNA
  expression vectors
• Use of DNA expression vector requires specific
  regulatory elements
• Judicious use of regulatory elements allows one
  to define the expression pattern
• Use homologous elements expressed in wt pattern
• Use heterologous elements expression in pattern
  of gene from which element came
• Use of constitutive heterologous elements (viral
  LTR or housekeeping gene drive expression
  everywhere in embryo

56
Biochemical AnalysisFunctional Cloning

• Cloning via DNA interaction or protein
  interaction
• Screen cDNA expression library with DNA probe to
  identify DNA binding proteins
• Screen by yeast two hybrid to identify
  protein-protein interactions

57
Biochemical Analysis

• Function of secreted morphogens by exposing whole
  embryo, explants or cultured cells to purified
  morphogen
• Determining differentially expressed genes by
  detecting differences in protein expression
• 2D SDS-PAGE
• Separate proteins based on isoelectric point then
  by size
• Compare proteins from two cell types to identify
  unique proteins
• Purify protein, sequence, reverse transcribe
  oligonucleotide, screen cDNA library

58
Biochemical Analysis Bioactivity
59
Biochemical Analysis 2D Gel
60
Biochemical Analysis 2D Gel
Compare 2D Gels of proteins form two cell types
Cell type A
Cell type B
61
Biochemical Analysis2D Gel
Overlay 2D gels to identify cell-type-specific
proteins